A critical roadblock in the development of an HIV-1 vaccine is our current inability to deliver HIV-1 antigens efficiently to the immune system and to prime predictable, high frequency immune responses in humans. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 have elicited potent immune responses in preclinical studies. However, the high frequency of anti-Ad5 immunity in the developing world will likely limit the immunogenicity and clinical utility of rAd5 vaccines. We therefore propose the development of novel rAd vector-based vaccines for HIV-1. We hypothesize that rAd vector-based vaccines derived from rare Ad serotypes and engineered for improved immunogenicity will prove significantly more immunogenic than rAd5 vaccines in rhesus monkeys with anti-Ad5 immunity. We further hypothesize that the optimal rAd vaccine regimen will be a heterologous prime-boost regimen involving two different serotype vectors that are both rare in human populations, engineered for optimal immunogenicity, derived from different Ad subfamilies, and distinct from Ad5. To investigate these hypotheses, we propose the following four Specific Aims: 1. To compare the immunogenicity of rAd5, rAd35, and capsid chimeric rAd5/rAd35 vectors in rhesus monkeys with anti-Ad5 immunity; 2. To assess the immunogenicity of heterologous rAd prime-boost regimens involving vectors derived from two different Ad subfamilies in mice; 3. To assess the immunogenicity and protective efficacy of the optimal heterologous rAd prime-boost regimen against an SIVmac251 challenge in rhesus monkeys with and without anti-Ad5 immunity; and 4. To determine the immunogenicity and protective efficacy of the optimal heterologous rAd prime- boost regimen delivered by either systemic or mucosal routes against repetitive, low-dose, mucosal SIVmac251 challenges in rhesus monkeys with anti-Ad5 immunity. The overall goal of these studies is to develop a novel heterologous rAd prime-boost regimen for HIV-1 that is highly immunogenic in the presence of anti-Ad5 immunity and that can be advanced rapidly into clinical vaccine trials in the developing world.